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TRAIL in the mandarin fish Siniperca chuatsi: gene and its apoptotic effect in HeLa cells
1Institute of Hydrobiology, Chinese Academy of Sciences, Hubei Province, People's Republic of China.
Abstract:
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359bp, encoding a 283-amino-acid protein. This deduced protein contains the Cys(231), a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPalp, NF-kappaB and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis.
Insights
The mandarin fish TRAIL gene (SCTRAIL) was cloned and characterized. Overexpression of SCTRAIL induces apoptosis in HeLa cells, highlighting its role as an apoptosis-inducing ligand.
Area of Science:
- Molecular Biology
- Immunology
- Aquaculture
Background:
- Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a crucial mediator of programmed cell death.
- Understanding TRAIL in fish species like mandarin fish (Siniperca chuatsi) is important for aquaculture and comparative immunology.
Purpose of the Study:
- To clone and characterize the TRAIL gene from mandarin fish (Siniperca chuatsi), named SCTRAIL.
- To investigate the gene structure, promoter elements, expression patterns, and functional role of SCTRAIL in apoptosis.
Main Methods:
- Gene cloning and sequencing to determine the full-length cDNA and protein sequence of SCTRAIL.
- Genomic DNA analysis (Southern blotting) and promoter region identification (genome walking).
- Gene expression analysis (RT-PCR, Western blotting) and functional assays in HeLa cells (FACS, DNA laddering).
Main Results:
- The SCTRAIL cDNA is 1359 bp, encoding a 283-amino-acid protein with conserved TRAIL domains.
- The SCTRAIL gene spans approximately 9kb with six exons and contains putative transcription factor binding sites in its promoter.
- SCTRAIL is constitutively expressed in various tissues and its overexpression induces apoptosis in HeLa cells.
Conclusions:
- The SCTRAIL gene is structurally conserved and expressed in mandarin fish.
- SCTRAIL functions as an apoptosis-inducing ligand, similar to its mammalian counterparts.
